Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Summary

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

Abstract

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph—mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

Introduction

Peptidoglycan (PG) is a defining characteristic of bacteria that serves to maintain cell morphology, while providing structural support for proteins and other cellular components^1,2. The backbone of PG is composed of alternating β-1,4-linked N-acetyl muramic acid (MurNAc) and N-acetyl glucosamine (GlcNAc)^1,2. Each MurNAc possesses a short peptide bound at the ᴅ-lactyl residue that can be crosslinked to adjacent disaccharide-linked peptides (Figure 1A,B). This crosslinking produ....

Protocol

1. Peptidoglycan sample preparation

1. Growth of bacterial cultures
   NOTE: The growth of the bacterial cultures will vary depending on the bacterial species and the growth conditions being examined. The experimental parameters to be tested will define the growth conditions.
   1. Grow bacterial cultures under growth conditions required for the bacterial strain and experimental design. Grow bacteria as triplicate cultures (biological replicates) i.e., three separate colonies per strai.......

Discussion

This protocol describes a method to purify peptidoglycan from bacterial cultures, process for LC-MS detection and analyze composition using bioinformatic techniques. Here, we focus on Gram-negative bacteria and some slight modification will be required to enable analysis of Gram-positive bacteria.

The preparation of muropeptides has remained virtually the same since it was first produced in the 1960s^{9,11,15.......}

Materials

Name	Company	Catalog Number	Comments
Equipment
C18 reverse phase column - AdvanceBio Peptide column (100 mm x 2.1 mm 2.7 µm)	Agilent		LC-MS data acquisition
Heating mantle controller, Optichem	Fisher	50-401-788	for 4% SDS boil
Heating Mantle, 1000mL Hemispherical	Fisher	CG1000008	for 4% SDS boil
Incubator, 37°C			for sacculi purification and MS sample prep
Leibig condenser, 300MM 24/40,	Fisher	CG121805	for 4% SDS boil
Lyophilizer	Labconco		for lyophilization of sacculi
Magentic stirrer	Fisher	90-691-18	for 4% SDS boil
mass spectrometer Q-Tof model UHD 6530	Aglient		LC-MS data acquisition
microcentrifuge filters, Nanosep MF 0.2 µm	Fisher	50-197-9573	cleanup of sample before MS injection
Retort stand	Fisher	12-000-102	for 4% SDS boil
Retort clamp	Fisher	S02629	for 4% SDS boil
round bottom flask - 1 liter pyrex	Fisher	07-250-084	for 4% SDS boil
Sonicator model 120	Fisher	FB120	for sacculi purification
Sonicator - micro tip	Fisher	FB4422	for sacculi purification
Ultracentrifuge	Beckman		sacculi wash steps
Ultracentrifuge bottles, Ti45	Fisher	NC9691797	sacculi wash steps
Water supply	City		for water cooled condenser
Software
Chemdraw	Cambridgesoft		molecular editor for muropeptide fragmentation prediction
Excel	Microsoft		viewing lists of annotated muropeptides, abundance, isotopic patterns, etc.
MassHunter Acquisition	Aglient		running QTOF instrument
MassHunter Mass Profiler Professional	Aglient		bioinformatic differential analysis
MassHunter Personal Compound Database and Library Manager	Aglient		muropeptide m/z MS database
MassHunter Profinder	Aglient		recursive feature extraction
MassHunter Qualitative analysis	Aglient		viewing MS and MS/MS chromatograms
Prism	Graphpad		Graphing software
Perseus	Max Plank Institute of Biochemistry		1D annotation
Material
Acetonitrile	Fisher	A998-4
Ammonium acetate	Fisher	A637
Amylase	Sigma-Aldrich	A6380
Boric acid	Fisher	BP168-1
DNase	Fisher	EN0521
Formic acid	Sigma-Aldrich	27001-500ML-R
LC-MS tuning mix - HP0321	Agilent	G1969-85000
Magnesium chloride	Sigma-Aldrich	M8266
Magnesium sulfate	Sigma-Aldrich	M7506
Mutanolysin from Streptomyces globisporus ATCC 21553	Sigma-Aldrich	M9901
Nitrogen gas (>99% purity)	Praxair	NI 5.0UH-T
Phosphoric acid	Fisher	A242
Pronase E from Streptomyces griseus	Sigma-Aldrich	P5147
RNase	Fisher	EN0531
Sodium azide	Fisher	S0489
Sodium borohydride	Sigma-Aldrich	452890
Sodium dodecyl sulfate (SDS)	Fisher	BP166
Sodium hydroxide	Fisher	S318
Sodium Phosphate (dibasic)	Fisher	S373
Sodium Phosphate (monobasic)	Fisher	S369
Stains-all	Sigma-Aldrich	E9379